Method for performing fluorescent protein binding assay employing novel alkyl substituted fluorescent compounds and conjugates

ABSTRACT

Fluorescent antigen conjugates are provided comprising antigens covalantly bonded to at least one 2,7-dialiphatic substituted-9-phenyl-6-hydroxy-3H-xanthen-3-one, wherein the 1- and 8-positions are unsubstituted. Also provided are novel fluorescent compounds absorbing at wavelengths in excess of 500 nm, having active functionalities for linking to the antigen. Finally, methods are provided for analyzing antigens in serum, whereby serum interference is avoided.

This application is a division of application Ser. No. 399,506, filedJuly 19, 1982, now U.S. Pat. No. 4,481,136, which is a division ofapplication Ser. No. 73,158, filed Sept. 7, 1979, now U.S. Pat. No.4,351,760.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Fluorescent compounds find a wide variety of applications. They find usein fluorescent immunoassays, histochemical staining, displays, inks, andthe like. Of particular interest for the subject invention is the use ofantigenic conjugates (includes receptor conjugates) with fluorescentcompounds to be used in the determination of a variety of ligands, bothantigens and receptors. A substantial proportion of the ligands areassayed in physiological fluids, such as serum, where the serum canprovide substantial background fluorescence. One way to diminish thebackground fluorescence resulting from naturally present fluorescers isto provide a fluorescent compound which absorbs at relatively longwavelengths. The compound should desirably have a large Stokes shift, bestable under conditions of the assay, be relatively free of non-specificinterference, both from materials in solution and the compound to whichthe fluorescer is conjugated and to provide high quantum yields. Inaddition, for certain applications, it is desirable that the fluorescerbe coupled with a quencher molecule, that is a molecule which is capableof absorbing the energy of the fluorescer in the excited state whenwithin a predetermined distance, so that the fluorescer does notfluoresce.

2. Description of the Prior Art

A large number of fluorescein derivatives have been reported in theliterature. The following are believed to be the most exemplary inrelation to the subject invention and are reported in conjunction withthe Chemical Abstracts citation. The numbering is based on the parentmolecule 3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-(9H)xanthen]-3-one.

2',7'-di(n-hexyl) or di(n-heptyl)-4', 5'-dibromo-4,7-dichloro- arereported as being prepared, C.A. 31, 1621; 2',7'-di(n-hexyl)-, C.A. 31,1621; 2',7'-di(alkyl)-; C.A. 31, 1388; 2',7'-diethyl or 2',7'-dibutyl-,C.A. 27, 5056; 2',7'-dimethyl-, C.A. 83, 18972s;2',4',5',7'-tetrabromo-5 or 6-carboxy, C.A. 63, 13210h.

SUMMARY OF THE INVENTION

The subject compounds include novel fluorescent conjugates with membersof specific binding pairs, ligands and receptors, as well as thefluorescent precursors to the conjugates. The conjugates find a widevariety of uses, particularly as reagents in immunoassays. The compoundsare 2,7-dialiphatic-6-hydroxy-3H-xanthen-3-ones, normally having a leasttwo chloro substituents, with the precursors having a linking group orfunctionality on a group, either aliphatic or aromatic, bonded to the 2-or 9-position of the xanthene.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

The subject invention concerns fluorescent compounds, which are analogsof fluorescein, being particularly 2,7-dialiphaticsubstituted-9-substituted-6-hydroxy-3H-xanthen-3-ones, usually having aleast two chloro substituents at other than the 1,8-positions and havinga functional group for linking to a member of a specific binding pairbonded to a hydrocarbon group substituted at the 2- or 9-position,particularly 9-position, as well as the conjugates of the fluorescentcompound to the member of the specific binding pair. The conjugates findparticular use as reagents in assays for members of specific bindingpairs.

The fluorescent precursors will have at least about 15 carbon atoms,usually 21 carbon atoms, and usually not more than about 40 carbonatoms, usually having from about 22 to 36 carbon atoms. There willpreferably be at least two chlorine groups at other than the1,8-positions and may be as many as 6 chlorines. In addition tochlorine, the only other heteroatoms are bromine, chalcogen,particularly oxygen and sulfur, and nitrogen, there being at least 4heteroatoms and usually not more than 20 heteroatoms, more usually notmore than about 16 heteroatoms and preferably not more than about 12heteroatoms. Of the heteroatoms other than chlorine, there will be atleast 3 oxygens, more usually at least 5 oxygens, and other than theoxygens which are part of the xanthene chromophore, are oxygens asnon-oxo-carbonyl or oxy, particularly acid, ester or ether (normallybonded solely to carbon and hydrogen); sulfur is normally present assulfonyl, thioether or mercapto; while nitrogen is normally present asamino or amido (bonded solely to carbon and hydrogen).

The fluorescent compounds are further characterized by having absorptionmaxima in 0.05M phosphate buffer pH8 of at least about 500 nm, anextinction coefficient in the same medium of at least about 65,000, moreusually at least 70,000 and a Stokes shift in the same medium of atleast about 10 nm, more usually at least about 12 nm.

The 9-substituted-2,7-dialkylsubstituted xanthenes of this inventionwill for the most part have the following formula: ##STR1## wherein:

ρ is an aliphatic group, normally aliphatic hydrocarbylene (composedsolely of carbon and hydrogen), saturated or unsaturated, branched orstraight chain, particularly alkylene, more particularly (CH₂).sub.ε,wherein ε is of from 1 to 12, usually 1 to 6, more usually 1 to 4; ρ isnormally of from 1 to 12, usually 1 to 6, more usually 1 to 4 carbonatoms;

the two Ψ's are the same or different, normally being the same, exceptwhen linking to α, and are hydrogen, a non-oxo-carbonyl functionality orone of the Ψ's may be a non-oxo-carbonyl linking functionality;

L is a bond or divalent radical, usually an organic radical, of at leastone carbon atom and not more than 20, usually not more than 16, moreusually not more than 10 carbon atoms, normally having an aliphatic oraromatic hydrocarbon chain, or combination thereof, wherein thealiphatic chain is usually of from about 2 to 6 carbon atoms and thearomatic chain is of from about 6 to 12, usually 6 to 10 carbon atoms; Lnormally has from 0 to 4, when aromatic, usually 1 to 4, more usually 2to 4 substituents, wherein the substituents may be halo, particularlychloro; non-oxo-carbonyl; thio, including inert sulfur acids, esters andamides; amino, particularly tert-amino of amido; and oxy, wherein thesubstituents are normally of from 0 to 4 carbon atoms, there being atleast two carbon atoms between heteroatoms bonded to saturated carbonatoms;

α is an organic compound, a member of a specific binding pair, either aligand or receptor;

β is 1, when α is covalently bonded to Ψ or Ψ', and is otherwise 0; thecovalent bond normally involves an amido, methylene sec-amino, ether,thioether or azo link;

Ψ is a group terminating in a heteroatom containing functionality whennot bonded to α, wherein the terminal heteroatom containingfunctionality may be bonded directly to a carbon atom of L or through anoligomer of from 1 to 4 units, each unit of 1 to 4, usually 2 to 4carbon atoms, which units are amino acids, alkyleneamino, or alkyleneoxygroups; the terminal functionality is normally oxo, includingoxo-carbonyl and non-oxo-carbonyl; amino; oxy; thio; or active halogen;particularly non-oxo-carbonyl; and

ν is one when β is 0 and is otherwise on the average at least one andnot more than the molecular weight of α divided by 500, usually dividedby 1,000.

Desirably, there are from 2 to 6 chloro substituents on the fluorescentgroup (in the brackets), bonded at other than the 1,8-positions of thexanthenone. Also, the 4,5-positions may be unsubstituted or one or both,usually both, substituted with bromo, chloro, or alkyl of from 1 to 6,usually 1 to 3 carbon atoms.

The fluorescer compound or conjugate with the organic compound (α) maybe linked, covalently or noncovalently to a support. The conjugate maybe bound either through the fluoroescer or organic compound. The supportwill be described in greater detail subsequently.

For the most part, the compounds of this invention having a 9-phenylwill have the following formula: ##STR2## wherein:

R is an aliphatic group of from 1 to 8, usually 1 to 6, more usually 1to 4, and preferably 1 to 3 carbon atoms, which may be substituted orunsubstituted, aliphatically saturated or unsaturated, particularlyalkyl or carboxyalkyl of from 1 to 6, usually 1 to 4 carbon atoms;

Z is carboxy;

W is a bond or divalent radical having from 0 to 16, either 0 or usually1 to 16 carbon atoms, more usually 1 to 8 carbon atoms and from 0 to 10,usually 2 to 8 heteroatoms, which are chalcogen (oxygen and sulfur) ornitrogen, wherein chalcogen is present bonded solely to carbon (oxy oroxo) and nitrogen is present bonded solely to carbon and hydrogen (aminoand amido); carbon is normally aromatic or aliphatic, particularly freeof aliphatic unsaturation, having from 0 to 2 sites of ethylenicunsaturation; W is conveniently a monomer or oligomer of units of from 1to 4 carbon atoms e.g. alkylene, aminoacid, oxyalkylene, aminoalkylene,etc.;

Y may be taken together with A to form an active functionality capableof forming a covalent bond with a heterofunctionality, such as amino,hydroxy, mercapto; that is with those functionalities present on A, whenA is not taken together with Y, such as oxo, oxo- and non-oxo-carbonyl,oxy, thio, amino, active halo, active olefin, inorganic acyl group, e.g.sulfonyl, etc. or acts as a linking functionality, being eithermethylene or heteroatom containing;

A, when not taken together with Y, is a member of a specific bindingpair, which is ligand or receptor, wherein the ligand may be haptenic orantigenic, normally being of from about 125 molecular weight to anindefinite upper limit, although for the most part, most ligands will beunder 10 million molecular weight, more usually under 2 millionmolecular weight, with varying ranges depending upon the nature of theligand or receptor;

m will be 0 to 3, more usually 0 to 2; and

n will be 1 when Y and A are taken together and will otherwise be on theaverage 1 to the molecular weight of A divided by 500, more usuallydivided by 1,000, and more frequently divided by 2,000, wherein withspecific binding pair members over 600,000 molecular weight A willnormally be not greater than A divided by 5,000. In addition, there willusually be at least two chloro substituents bonded on any of theavailable positions where no specific atom is indicated. Also, the4,5-positions may be substituted as described previously. Furthermore,either the conjugate or the fluorescer precursor may be bonded to asupport of at least about 10,000 molecular weight and up to anindefinite molecular weight.

A preferred group of compounds will for the most part have the followingformula: ##STR3## wherein:

R' is alkylene of from 1 to 6, usually 1 to 4, and preferably 1 to 3carbon atoms;

D is hydrogen or carboxy;

Z' is carboxy;

m' is 0 to 3, usually 0 to 2;

Y' may be taken together with A' to form an active functionally whichmay be non-oxo-carbonyl, including the sulfur analog thereof, aminobonded to at least one hydrogen atom, mercapto, active ethylene, usuallyhaving an α-carbonyl, halomethylcarbonyl, wherein halo is of atomicnumber 17 to 53, sulfonyl, or the like; when not taken together with A',Y' will be a linking functionality, either methylene or a heteroatomcontaining linking functionality, usually being an amide, ester, etheror azo link;

W' is a bond or linking group of from 1 to 16, usually 1 to 12, andpreferably 1 to 8 atoms other than hydrogen, which are carbon, nitrogen,oxygen or sulfur, preferably carbon, nitrogen and oxygen, there beingfrom 0 to 8 carbon atoms and 0 to 8 heteroatoms, with the number ofcarbon atoms and heteroatoms being at least 1, wherein nitrogen will bebonded solely to hydrogen and carbon, and will be either amino or amido,oxygen nd sulfur will be bonded solely to carbon as oxy (thio) or oxo(thiono) and carbon is normally aliphatic and usually free of aliphaticunsaturation, generally having from 0 to 1 site of ethylenicunsaturation; W' may be alkylene or alkenylene of from 1 to 8, usually 1to 4 carbon atoms, oxoalkylene or oxoalkenylene of from 1 to 8, usually1 to 4 carbon atoms, imino (NH), N-formyl amino acid or N-formylpoly(amino acid) e.g. glycine or polyglycine, there being from about 1to 4 amino acids, with the terminal carboxy being Y'A', or the like;

n' is 1 when Y' and A' are taken together and otherwise is on theaverage at least 1 to the molecular weight of A' divided by 500, usuallydivided by 1,000, more usually divided by 2,000, and when A' is over500,000 molecular weight, more usually divided by 5,000;

there generally being not more than 5 carboxyl groups, usually not morethan 4 carboxyl groups in total, and there being from 0 to 6 chlorogroups, preferably 2 to 5 chloro groups bonded to available carbonatoms; and

A' is a member of a specific binding pair, a ligand or receptor, whereinthe ligand may be haptenic or antigenic, and haptenic ligands willinclude compounds of interest such as drugs, hormones, pollutants,compounds of interest in processing, agricultural chemicals,metabolites, and the like;

antigens will primarily be proteins, polysaccharides or nucleic acids,individually or in combination with each other or other materials, suchas in cells, viruses, phage, or the like. The haptens will normally befrom about 125 to 2,000, more usually to 1,000 molecular weight, whilethe antigens will normally be from about 2,000, more usually 5,000molecular weight up to an indefinite molecular weight, usually notexceeding 10 million, more usually not exceeding 2 million.

The 4,5-positions are preferably unsubstituted or chloro-substituted.

In addition, the above conjugate may be bonded to a support. Varioussupports may be employed, both soluble or insoluble, swellable ornonswellable, by aqueous or organic solvents, naturally occurring orsynthetic, organic or inorganic, porous or nonporous, or the like.Various polymeric materials include vinyl polymers and copolymers,polysaccharides, silicones, glass, carbon particles, such as graphite orcharcoal, metals or metal compounds, poly(amino acids), nucleic acids orthe like.

For the most part, the fluorescent compounds of the subject inventionemployed for conjugation will have the following formula: ##STR4##wherein:

R¹ is alkylene of from 1 to 6, usually 1 to 4, preferably 1 to 2 carbonatoms;

D¹ is hydrogen or carboxy, preferably hydrogen;

Z¹ is carboxy;

E^(a) is hydrogen, alkyl of from 1 to 6, usually 1 to 3 carbon atoms, orchloro;

E¹ is chloro;

W¹ is a bond or linking group of from 1 to 12, usually 1 to 8 atomsother than hydrogen, and generally 1 to 8, usually 1 to 6 atoms in thechain wherein the atoms are carbon, nitrogen, oxygen and sulfur,particularly carbon, nitrogen and oxygen, wherein the carbon isaliphatic, the nitrogen is present as amido or amino, particularly aminobonded solely to carbon, and oxygen and sulfur are bonded solely tocarbon and are oxy or oxo or the sulfur analogs thereof:

W¹ will generally be aliphatic, being saturated or unsaturated, normallysaturated, having from 0 to 1 site of ethylenic unsaturation, alkyleneor alkenylene of from 1 to 8, usually 1 to 4 carbon atoms, N-formylamino acid of N-formyl poly(amino acid), where the terminal carboxy isderived from Y¹ A¹, amino, mercapto, or the like;

Y¹ A¹ are taken together to form a functionality for linking, wherein Y¹A¹ are bonded solely to carbon or nitrogen, with the proviso that whenY¹ and A¹ are bonded to nitrogen, Y¹ A¹ are carbonyl, including thenitrogen and sulfur analogs thereof and can be doubly bonded tonitrogen;

Y¹ A¹ can be non-oxo-carbonyl, haloacetyl, halogen of atomic no. 9 to53, particularly chloro or bromo, maleimido, mercapto, amino, orinorganic acyl, having phosphorous or sulfur as the central atom;

m¹ is 0 to 3, usually 0 to 2, there usually being not more than a totalof 5 carboxyl groups in the molecule, usually not more than a total of 4carboxyl groups, and preferably not more than about 2 carboxyl groups,other than Y¹ A¹ ;

x is 0 to 4, preferably 2 to 4, there generally being not more than atotal of 6 chloro groups in the molecule, usually not more than a totalof 4 chloro groups, wherein x plus m¹ is not greater than 4.

For the most part, the compositions of this invention when bonded toligand or support will have the following formula: ##STR5## wherein:

E^(b) is hydrogen or chloro;

E² is chloro;

Z² is carboxy;

R² is alkylene of from 1 to 6, usually 1 to 3, preferably 1 to 2 carbonatoms;

D² is hydrogen or carboxy, preferably hydrogen;

W² is a bond or linking chain, when a linking chain being of from 1 to12, usually of from 1 to 10, and preferably of from about 1 to 8 atomsother than hydrogen, having from about 1 to 10, usually from about 1 to8, and preferably from about 1 to 6 atoms in the chain or spacer arm,wherein the atoms are carbon, oxygen, nitrogen and sulfur, particularlycarbon, oxygen and nitrogen in the spacer arm, wherein oxygen and sulfurare bonded solely to carbon, as oxy or oxo, and nitrogen is bondedsolely to carbon and hydrogen, namely amino and amido, whereinheteroatoms bonded to saturated carbon atoms are separated by at leasttwo carbon atoms;

W² is particularly alkylene, carboxamidoalkylene, wherein alkylene is offrom about 1 to 2 carbon atoms(--CONHC₁₋₂ --)_(a), wherein a is in therange of from about 1 to 4, usually 1 to 3;

Y² is non-oxo-carbonyl, carbamyl, thiocarbamyl, methylene, amino, orthio, particularly a functionality having a non-oxo-carbonyl group orsulfur analog thereof;

x² is 0 to 4;

m² is 0 to 3, preferably 1 to 2;

n² is 1 to the molecular weight of A² divided by 500, usually divided by1,000, more usually divided by 2,000, wherein when A² is a ligand ofbetween about 125 to 2,000 molecular weight, n² will generally be offrom about 1 to 20, when A² is a ligand of from about 2,000 to 600,000molecular weight, n² will generally be in the range of about 1 to 100,more usually in the range of about 2 to 50; and

A² is a ligand of at least about 125 molecular weight and may be 10million or more molecular weight, which is haptenic or antigenic,wherein haptens are from about 125 to 2,000 molecular weight andantigens will generally range from about 5,000 to 10 million molecularweight, more usually from about 5,000 to 2 million molecular weight andfrequently from about 5,000 to 600,000 molecular weight, the ligandbeing a member of a specific binding pair, which comprises a compoundhaving at least 1 determinant or epitopic site and a receptor which iscapable of recognizing the determinant size or A² is a receptor of fromabout 10,000 to 1 million molecular weight.

Finally, in some instances it may be desirable to have the fluorescentcompound or the conjugate of the fluorescent compound with ligand,bonded to a support, where the linkage may be derived from either thefluorescent compound or the ligand, normally the ligand. In thissituation, the linking group may be any convenient functionality whichis present on the fluorescent compound or the ligand or a functionalitywhich may be introduced, particularly on the ligand. These compositionswill for the most part have the following formula, where the symbols arederived from the previous formula for the conjugate for the most part:##STR6## wherein:

all of the symbols have been defined previously, except for:

n³ which is at least 1 and up to the molecular weight of A² divided by500, usually 1000, more usually 1,500, with the proviso that when q is0, n³ is 1;

q which is 0 or 1;

p which is at least 1 and of up to the molecular weight of the supportdivided by 500, more usually the molecular weight of the support dividedby 1,000, wherein when the molecular weight of the support exceeds500,000, p will normally be not greater than the molecular weight of thesupport divided by 5,000, more usually divided by 10,000; and

Support intends a macromolecular support of at least about 10,000molecular weight, which may be naturally occurring or synthetic, havinga plurality of functionalities for linking e.g. carboxy, hydroxy, oramino, usually being a polymer, such as a polysaccharide or an additionpolymer; the support being bonded to the conjugate by any convenientfunctionality remaining an A² or the conjugate in the brackets, theparticular manner of linking not being a significant aspect of thesubject invention. For example, if A² is a poly(amino acid), carboxylicgroups on the support can be used for amide formation or maleimidegroups may be introduced and linked to mercapto groups.

Quite obviously, the compounds of the subject invention can be modifiedso as not to be within the above formulas, without significantlyaffecting the properties of the compounds. For example, one or more ofthe acidic anionic groups could be esterified or amidified, or alkylgroups can be substituted on the phenyl, as well as other groups, suchas cyano, nitro, or the like. However, these changes will in most casesrequire additional synthetic steps which are not warranted by the degreeof enhancement, if any, in the spectroscopic or chemical properties ofthe resulting product.

Turning now to a consideration of the individual components of thesubject compositions, the fluorescein derivatives will be consideredfirst. The following is a list of illustrative fluorescein derivativescoming within the scope of the subject invention.

TABLE I

2,7-dimethyl-4,5-dichloro-9-(2',4',5'-tricarboxyphenyl)-6-hydroxy-3-xanthen-3-one

2,7-diethyl-4,5-dichloro-9-(2',4',5'-tricarboxy-3',6'-dichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dihexyl-9-(2',4',5'-tricarboxyphenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-4,5-dichloro-9-(2'-carboxy-4'-isothiocyanato-3',5'-dichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-9-(2'-carboxy-4'-isocyanato-3',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-9-(4'-carboxy-5'-carboxylphenyl)-glycylglycylglycineamide-6-hydroxy-3H-xanthen-3-one

2,7-di(carboxymethyl)-9-(4',5',-dicarboxy-2',3',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-di(carboxypropyl)-4,5-dichloro-9-(3',4'-dicarboxyphenyl)-6-hydroxy-3H-xanthen-3-one

2,7-diethyl-9-(2'-carboxy-4'-amino-3',5'-dichlorophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-9-(2'-carboxy-4'-mercaptophenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-9-(2'-carboxy-4'-carboxymethylphenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-9-(2'-carboxy-4'-(4"-carboxybutyl)-phenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-4,5-dichloro-9-(2',4'-dicarboxy-5'-(carboxamidomethylene)phenyl)-6-hydroxy-3H-xanthen-3-one

2,7-dimethyl-4,5-dichloro-9(3'-carboxypropyl)-6-hydroxy-3H-xanthen-3-one.

As indicated previously, the fluorescein derivatives of the subjectinvention will be conjugated with ligands and/or supports. The followingis a description of the applicable ligands.

Analyte

The ligand analytes of this invention are characterized by beingmonoepitopic or polyepitopic. The polyepitopic ligand analytes willnormally be poly(amino acids) i.e. polypeptides and proteins,polysaccharides, nucleic acids, and combinations thereof. Suchcombinations of assemblages include bacteria, viruses, chromosomes,genes, mitochondria, nuclei, cell membranes, and the like.

For the most part, the polyepitopic ligand analytes employed in thesubject invention will have a molecular weight of at least about 5,000,more usually at least about 10,000. In the poly(amino acid) category,the poly(amino acids) of interest will generally be from about 5,000 to5,000,000 molecular weight, more usually from about 20,000 to 1,000,000molecular weight; among the hormones of interest, the molecular weightswill usually range from about 5,000 to 60,000 molecular weight.

The wide variety of proteins may be considered as to the family ofproteins having similar structural features, proteins having particularbiological functions, proteins related to specific microorganisms,particularly disease causing microorganisms, etc.

The following are classes of proteins related by structure:

protamines

histones

albumins

globulins

scleroproteins

phosphoproteins

mucoproteins

chromoproteins

lipoproteins

nucleoproteins

glycoproteins

proteoglycans

unclassified proteins, e.g. somatotropin,

prolactin, insulin, pepsin

A number of proteins found in the human plasma are important clinicallyand include:

Prealbumin

Albumin

α₁ -Lipoprotein

α₁ -Acid glycoprotein

α₁ -Antitrypsin

α₁ -Glycoprotein

Transcortin

4.6S-Postalbumin

Tryptophan-poor

α₁ -glycoprotein

α₁ X-Glycoprotein

Thyroxin-binding globulin

Inter-α-trypsin-inhibitor

Gc-globulin

(Gc 1-1)

(Gc 2-1)

(Gc 2-2)

Haptoglobin

(Hp 1-1)

(Hp 2-1)

(Hp 2-2)

Ceruloplasmin

Cholinesterase

α₂ -Lipoprotein(s)

Myoglobin

C-Reactive Protein

α₂ -Macroglobulin

α₂ -HS-glycoprotein

Zn-α₂ -glycoprotein

α₂ -Neuramino-glycoprotein

Erythropoietin

β-lipoprotein

Transferrin

Hemopexin

Fibrinogen

Plasminogen

β₂ -glycoprotein I

β₂ -glycoprotein II

Immunoglobulin G

(IgG) or γG-globulin

Mol. formula:

γ₂ κ₂ or γ₂ λ₂

Immunoglobulin A (IgA)

or γA-globulin

Mol. formula:

(α₂ κ₂)^(n) or (α₂ λ₂)^(n)

Immunoglobulin M

(IgM) or γM-globulin

Mol. formula:

(μ₂ μ₂)⁵ or (μ₂ λ₂)⁵

Immunoglobulin D(IgD)

or γD-Globulin (γD)

Mol. formula:

(δ₂ κ₂) or δ₂ λ₂)

Immunoglobulin E (IgE)

or γE-Globulin (γE)

Mol. formula:

(ε₂ κ₂) or (ε₂ λ₂)

Free κ and λ light chains

Complement factors:

C'1

C'1q

C'1r

C'1s

C'2

C'3

β₁ A

α₂ D

C'4

C'5

C'6

C'7

C'8

C'9

Important blood clotting factors include:

    ______________________________________                                        BLOOD CLOTTING FACTORS                                                        International                                                                 designation      Name                                                         ______________________________________                                        I                Fibrinogen                                                   II               Prothrombin                                                  IIa              Thrombin                                                     III              Tissue thromboplastin                                        V and VI         Proaccelerin, accelerator                                                     globulin                                                     VII              Proconvertin                                                 VIII             Antihemophilic globulin                                                       (AHG)                                                        IX               Christmas factor,                                                             plasma thromboplastin                                                         component (PTC)                                              X                Stuart-Prower factor,                                                         autoprothrombin III                                          XI               Plasma thromboplastin                                                         antecedent (PTA)                                             XII              Hagemann factor                                              XIII             Fibrin-stabilizing factor                                    ______________________________________                                    

Important protein hormones include:

Peptide and Protein Hormones

Parathyroid hormone

(parathromone)

Thyrocalcitonin

Insulin

Glucagon

Relaxin

Erythropoietin

Melanotropin

(melanocyte-stimulating

hormone; intermedin)

Somatotropin

(growth hormone)

Corticotropin

(adrenocorticotropic hormone)

Thyrotropin

Follicle-stimulating hormone

Luteinizing hormone

(interstitial cell-stimulating hormone)

Luteomammotropic hormone

(luteotropin, prolactin)

Gonadotropin

(chorionic gonadotropin)

Tissue Hormones

Secretin

Gastrin

Angiotensin I and II

Bradykinin

Human placental lactogen

Peptide Hormones from the Neurophypophysis

Oxytocin

Vasopressin

Releasing factors (RF)

CRF, LRF, TRF, Somatotropin-RF,

GRF, FSH-RF, PIF, MIF

Other polymeric materials of interest are mucopolysaccharides andpolysaccharides.

Illustrative antigenic polysaccharides derived from microorganisms areas follows:

    ______________________________________                                        Species of Microorganisms                                                                        Hemosensitin Found in                                      ______________________________________                                        Streptococcus pyogenes                                                                           Polysaccharide                                             Diplococcus pneumoniae                                                                           Polysaccharide                                             Neisseria meningitidis                                                                           Polysaccharide                                             Neisseria gonorrheae                                                                             Polysaccharide                                             Corynebacterium diptheriae                                                                       Polysaccharide                                             Actinobacillus mallei;                                                                           Crude extract                                              Actinobacillus whitemori                                                      Francisella tularensis                                                                           Lipopolysac-                                                                  charide                                                                       Polysaccharide                                             Pasteurella pestis                                                            Pasteurella pestis Polysaccharide                                             Pasteurella multocida                                                                            Capsular antigen                                           Brucella abortus   Crude extract                                              Haemophilus influenzae                                                                           Polysaccharide                                             Haemophilus pertussis                                                                            Crude                                                      Treponela reiteri  Polysaccharide                                             Veillonella        Lipopolysac-                                                                  charide                                                    Erysipelothrix     Polysaccharide                                             Listeria monocytogenes                                                                           Polysaccharide                                             Chromobacterium    Lipopolysac-                                                                  charide                                                    Mycobacterium tuberculosis                                                                       Saline extract of                                                             90% phenol                                                                    extracted                                                                     mycobacteria                                                                  and polysaccharide                                                            fraction of                                                                   cells and                                                                     turberculin                                                Klebsiella aerogenes                                                                             Polysaccharide                                             Klebsiella cloacae Polysaccharide                                             Salmonella typhosa Lipopolysac-                                                                  charide,                                                                      Polysaccharide                                             Salmonella typhi-murium;                                                                         Polysaccharide                                             Salmonella derby                                                              Salmonella pullorum                                                           Shigella dysenteriae                                                                             Polysaccharide                                             Shigella flexneri                                                             Shigella sonnei    Crude, Poly-                                                                  saccharide                                                 Rickettsiae        Crude extract                                              Candida albicans   Polysaccharide                                             Entamoeba histolytica                                                                            Crude extract                                              ______________________________________                                    

The microorganisms which are assayed may be intact, lysed, ground orotherwise fragmented, and the resulting composition or portion, e.g. byextraction, assayed. Microorganisms of interest include:

Corynebacteria

Corynebacterium diptheriae

Pneumococci

Diplococcus pneumoniae

Streptococci

Streptococcus pyogenes

Streptococcus salivarus

Staphylococci

Staphylococcus aureus

Staphylococcus albus

Neisseriae

Neisseria meningitidis

Neisseria gonorrheae

    ______________________________________                                        Enterobacteriaciae                                                            Escherichia coli                                                              Aerobacter aerogenes      The coliform                                        Klebsiella pneumoniae     bacteria                                            Salmonella typhosa                                                            Salmonella choleraesuis   The Salmonellae                                     Salmonella typhimurium                                                        Shigella dysenteriae                                                          Shigella schmitzii                                                            Shigella arabinotarda     The Shigellae                                       Shigella flexneri         The Shigellae                                       Shigella boydii                                                               Shigella Sonnei                                                               Other enteric bacilli                                                         Proteus vulgaris                                                              Proteus mirabilis         Proteus species                                     Proteus morgani                                                               Pseudomonas aeruginosa                                                        Alcaligenes faecalis                                                          Vibrio cholerae                                                               Hemophilus-Bordetella group                                                   Hemophilus influenzae,                                                                          H. ducreyi                                                                    H. hemophilus                                                                 H. aegypticus                                                                 H. parainfluenzae                                           Bordetella pertussis                                                          ______________________________________                                    

Pasteurellae

Pasteurella pestis

Pasteurella tulareusis

Brucellae

Brucella melitensis

Brucella abortus

Brucella suis

Aerobic Spore-forming Bacilli

Bacillus anthracis

Bacillus subtilis

Bacillus megaterium

Bacillus cereus

Anaerobic Spore-forming Bacilli

Clostridium botulinum

Clostridium tetani

Clostridium perfringens

Clostridium novyi

Clostridium septicum

Clostridium histolyticum

Clostridium tertium

Clostridium bifermentans

Clostridium sporogenes

Mycobacteria

Mycobacterium tuberculosis hominis

Mycobacterium bovis

Mycobacterium avium

Mycobacterium leprae

Mycobacterium paratuberculosis

Actinomycetes (fungus-like bacteria)

Actinomyces israelii

Actinomyces bovis

Actinomyces naeslundii

Nocardia asteroides

Nocardia brasiliensis

    ______________________________________                                        The Spirochetes                                                               ______________________________________                                        Treponema pallidum    Spirillum minus                                         Treponema pertenue    Streptobacillus                                                               moniliformis                                            Treponema carateum                                                            Borrelia recurrentis                                                          Leptospira icterohemorrhagiae                                                 Leptospira canicola                                                           ______________________________________                                    

Mycoplasmas

Mycoplasma pneumoniae

Other pathogens

Listeria monocytogenes

Erysipelothrix rhusiopathiae

Streptobacillus moniliformis

Donvania granulomatis

Bartonella bacilliformis

Rickettsiae (bacteria-like parasites)

Rickettsia prowazekii

Rickettsia mooseri

Rickettsia rickettsii

Rickettsia conori

Rickettsia australis

Rickettsia sibiricus

Rickettsia akari

Rickettsia tsutsugamushi

Rickettsia burnetii

Rickettsia quintana

Chlamydia (unclassifiable parasites bacterial/viral)

Chlamydia agents (naming uncertain)

Fungi

Cryptococcus neoformans

Blastomyces dermatidis

Histoplasma capsulatum

Coccidioides immitis

Paracoccidioides brasiliensis

Candida albicans

Aspergillus fumigatus

Mucor corymbifer (Absidia corymbifera)

    ______________________________________                                        Rhizopus oryzae                                                               Rhizopus arrhizus          Phycomycetes                                       Rhizopus nigricans                                                            ______________________________________                                    

Sporotrichum schenkii

Fonsecaea pedrosoi

Fonsecaea compacta

Fonsecaea dermatidis

Cladosporium carrionii

Phialophora verrucosa

Aspergillus nidulans

Mandurella mycetomi

Madurella grisea

Allescheria boydii

Phialosphora jeanselmei

Microsporum gypseum

Trichophyton mentagrophytes

Keratinomyces ajelloi

Microsporum canis

Trichophyton rubrum

Microsporum andouini

Viruses Adenoviruses Herpes Viruses

Herpes simplex

Varicella (Chicken pox)

Herpes Zoster (Shingles)

Virus B

Cytomegalovirus

Pox Viruses

Variola (smallpox)

Vaccinia

Poxvirus bovis

Paravaccinia

Molluscum contagiosum

Picornaviruses

Poliovirus

Coxsackievirus

Echoviruses

Rhinoviruses

Myxoviruses

Influenza (A, B, and C)

Parainfluenza (1-4)

Mumps Virus

Newcastle Disease Virus

Measles Virus

Rinderpest Virus

Canine Distemper Virus

Respiratory Syncytial Virus

Rubella Virus

Arboviruses

Eastern Equine Eucephalitis Virus

Western Equine Eucephalitis Virus

Sindbis Virus

Chikugunya Virus

Semliki Forest Virus

Mayora Virus

St. Louis Encephalitis Virus

California Encephalitis Virus

Colorada Tick Fever Virus

Yellow Fever Virus

Dengue Virus

Reoviruses

Reovirus Types 1-3

Hepatitis

Hepatitis A Virus

Hepatitis B Virus

Tumor Viruses

Rauscher Leukemia Virus

Gross Virus

Maloney Leukemia Virus

The monoepitopic ligand analytes will generally be from about 100 to2,000 molecular weight, more usually from 125 to 1,000 molecular weight.The analytes of interest include drugs, metabolites, pesticides,pollutants, and the like. Included among drugs of interest are thealkaloids. Among the alkaloids are morphine alkaloids, which includesmorphine, codeine, heroin, dextromethorphan, their derivatives andmetabolites; cocaine alkaloids, which includes cocaine and benzoylecgonine, their derivatives and metabolites; ergot alkaloids, whichincludes the diethylamide of lysergic acid; steroid alkaloids; iminazoylalkaloids; quinazoline alkaloids, isoquinoline alkaloids; quinolinealkaloids; which includes quinine and quinidine; diterpene alkaloids;their derivatives and metabolites.

The next group of drugs includes steroids, which includes the estrogens,gestogens, androgens, andrenocortical steroids, bile acids, cardiotonicglycosides and aglycones, which includes digoxin and digoxigenin,saponins and sapogenins, their derivatives and metabolites. Alsoincluded are the steroid mimetic substances, such as diethylstilbestrol.

The next group of drugs is lactams having from 5 to 6 annular members,which include the barbiturates, e.g. phenobarbital and secobarbital,diphenylhydantonin, primidone, ethosuximide, and their metabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes the amphetamines, catecholamines, whichincludes ephedrine, L-dopa, epinephrine, narceine, papaverine, theirmetabolites.

The next group of drugs is aminoalkylbenzenes, with alkyl of from 2 to 3carbon atoms, which includes ephedrine, L-dopa, epinephrine, narceine,papverine, their metabolites and derivatives.

The next group of drugs is benzheterocyclics which include oxazepam,chlorpromazine, tegretol, imipramine, their derivatives and metabolites,the heterocyclic rings being azepines, diazepines and phenothiazines.

The next group of drugs is purines, which includes theophylline,caffeine, their metabolites and derivatives.

The next group of drugs includes those derived from marijuana, whichincludes cannabinol and tetrahydrocannabinol.

The next group of drugs includes the vitamins such as A, B, e.g. B₁₂, C,D, E and K, folic acid, thiamine.

The next group of drugs is prostaglandins, which differ by the degreeand sites of hydroxylation and unsaturation.

The next group of drugs is antibiotics, which include penicillin,chloromycetin, actinomycetin, tetracycline, terramycin, theirmetabolites and derivatives.

The next group of drugs is the nucleosides and nucleotides, whichinclude ATP, NAD, FMN, adenosine, guanosine, thymidine, and cytidinewith their appropriate sugar and phosphate substituents.

The next group of drugs is miscellaneous individual drugs which includemethadone, meprobamate, serotonin, meperidine, amitriptyline,nortriptyline, lidocaine, procaineamide, acetylprocaineamide,propranolol, griseofulvin, valproic acid, butyrophenones,antihistamines, anticholinergic drugs, such as atropine, theirmetabolites and derivatives.

The next group of compounds is amino acids and small peptides whichinclude polyiodothyronines e.g. thyroxine, and triiodothyronine,oxytocin, ACTH, angiotensin, met-and leu-enkephalin their metabolitesand derivatives.

Metabolites related to diseased states include spermine, galactose,phenylpyruvic acid, and porphyrin Type 1.

The next group of drugs is aminoglycosides, such as gentamicin,kanamicin, tobramycin, and amikacin.

Among pesticides of interest are polyhalogenated biphenyls, phosphateesters, thiophosphates, carbamates, polyhalogenated sulfenamides, theirmetabolites and derivatives.

For receptor analytes, the molecular weights will generally range from10,000 to 2×10⁶, more usually from 10,000 to 10⁶. For immunoglobulins,IgA, IgG, IgE and IgM, the molecular weights will generally vary fromabout 160,000 to about 10⁶. Enzymes will normally range from about10,000 to 6000,000 in molecular weight. Natural receptors vary widely,generally being at least about 25,000 molecular weight and may be 10⁶ orhigher molecular weight, including such materials as avidin, thyroxinebinding globulin, thyroxine binding prealbumin, transcortin, etc.

In many applications for the subject fluorescein derivatives, it will bedesirable to have the ligand bonded to a support, either directly,through the intermediacy of a ligand, or directly to the support, whilebound to a ligand.

A wide variety of supports may be employed. The particles or supportscan be derived from naturally occurring materials, naturally occurringmaterials which are synthetically modified and synthetic materials. Ofparticular interest are polysaccharides, particularly crosslinkedpolysaccharides, such as agarose, which is available as Sepharose,dextran, available as Sephadex and Sephacyl, cellulose, starch, and thelike. Other materials include polyacrylamides, polystyrene, polyvinylalcohol, copolymers of hydroxyethyl methacrylate and methylmethacrylate, silicones, glasses, available as Bioglas, nucleic acids,poly(amino acids), cells or the like. In addition to solid particles,liquid particles may also be employed having a lipophilic or amphiphilicmembrane, which serves to contain an internal fluid and define a space.Such particles include vesicles, cells and liposomes.

The particles may be porous or nonporous, swellable or nonswellable byaqueous or organic media, normally having a variety of functionalities,such as hydroxyl, amino or carboxy, either bonded directly to thebackbone or by means of a spacer arm, crosslinked or noncrosslinked,smooth or rough surface, or the like.

The porous particles may have a wide variety of cut off sizes, generallyvarying from about 10,000 to many million molecular weight, usually notexceeding 20 million molecular weight.

As already indicated, a wide variety of linking chains may be employedbetween the fluorescein compound and the ligand and/or support. Thechoice of linking group will vary widely, depending upon the availablefunctionalities or functionalities which may be readily introduced, thedesired length of the linking arm, the desirabilty of having the linkingarm provide for a particular environment, chemical property or physicalproperty, e.g. positively or negatively charged, solubility enhancement,dipole effects, or the like.

The following table indicates a variety of linking groups which may beemployed for linking the fluorescein compound to the ligand:

                  TABLE II                                                        ______________________________________                                        Fluorescein                   Ligand                                          functionality                                                                           Linking group       functionality                                   ______________________________________                                        CO.sub.2 H                                                                              (NHGCO).sub.g       NH.sub.2                                        CO.sub.2 H or                                                                           (NHG'NH).sub.g' NHG'NH                                                                            CO.sub.2 H                                      SO.sub.3 H                                                                              NHG'N(CH.sub.2 CH.sub.2).sub.2 NG'NH                                SH                                                                                       ##STR7##           CO.sub.2 H                                       COCH.sub.2 halo                                                                         ##STR8##            CO.sub.2 H                                     NH.sub.2  COGCO               NH.sub.2                                        ______________________________________                                    

wherein:

G is alkylene of from 1 to 8, usually 1 to 6 carbon atoms, G' isalkylene of from 2 to 6, usually 2 to 4 carbon atoms, and g and g' are 1to 6, usually 1 to 4. It is understood that the above table is merelyillustrative of the more common linking groups, other linking groupsbeing available in special situations. For example, where phenolicgroups are present, such as tyrosyl, aryl diazonium functionalities maybe employed. Furthermore, it is understood that the functionalities forthe fluorescein and ligand may be reversed, with concomitant reversal ofthe direction of the linking group.

The subject compounds have many desirable properties. The products havesignificant water solubility which allow them to be conjugated to a widevariety of polypeptides, without significantly adversely affecting thewater solubility of the polypeptide, nor having the polypeptideadversely affect the spectroscopic properties of the subject compounds.

As for the spectroscopic properties of the compounds, the compoundsabsorb at relatively long wavelengths, generally in excess of 500 nm,more usually in excess of 510 nm. Thus, naturally occurring fluorescencewhich may be encountered when working with physiological fluids issubstantially avoided by employing exciting light at a wavelength rangewhich does not significantly excite the naturally occurring fluorescers.In addition, the compounds have relatively sharp absorption peaks andemission peaks. Because of this, efficient overlap can be obtainedbetween fluorescers and quenchers which allow for efficient quenching upto distances of about 70 Å. The fluorescing compounds also have largeStokes shifts, so that the absorption band and emission band peaks areseparated by at least 10 nm, frequently by at least 15 nm. The largeStokes shifts minimize background interference with the observedfluorescence.

The compounds of the subject invention are prepared in accordance withconventional means. The appropriate resorcinol and carboxylic acid oranhydride are combined in the presence of a Lewis acid e.g. zincchloride, and the mixture heated at an elevated temperature for asufficient time to provide the desired product. The product may then bepurified by conventional means.

The subject compounds find a wide variety of applications, particularlyfor use as conjugates to ligands and/or supports in protein bindingassays. The conjugates can be used for determining qualitatively,semi-quantitatively or quantitatively the presence of a compound ofinterest in a sample. Where compounds are to be detected inphysiological fluids, the fluids may include serum, urine, saliva, lymphor the like. Where the compound of interest is involved in chemicalprocessing or ecological concerns, the sample may involve an aqueousmedium, an organic medium, soil, inorganic mixtures, or the like.

For use in immunoassays or in other diagnostic situations, thespectroscopically active compounds of this invention will be conjugatedto a compound of interest, including a receptor for an analyte or aligand. (By receptor is intended any molecule which specifically bindsto a spatial and polar molecular organization, while a ligand is anorganic molecule having such organization.) The analyte will normally behaptenic or antigenic. Where these compounds do not have availablefunctionalities for linking, they will be modified to introduce such afunctionality, while still retaining the immunological properties in theresulting product. Those compounds which are analogs of the analyte,which analyte may also be referred to as a ligand, will be referred toas ligand analogs.

As indicated previously, the compounds of this invention may beconjugated to compounds which may be measured by known immunoassaytechniques. The resulting conjugates are reagents which compete in anassay medium for the compound of interest or analyte in a sample.Therefore, the conjugate retains a sufficient proportion of thestructure of the compound of interest to be able to compete with thecompound of interest for receptor, usually an antibody.

The analytes or their analogs, receptors or ligands, which areconjugated to the spectroscopically active compounds of this inventionare characterized by being monoepitopic or polyepitopic.

The assays will normally involve a change of spectroscopic propertiesdue to a change in environment about the spectroscopically activecompound or the bringing together of a fluorescer-quencher pair withinsufficient proximity for the quencher to interact with the fluorescer.Alternatively, methods can be employed which involve the separation ofassociated and unassociated fluorescer and the detection of thefluorescer in one or both of the fractions.

In a first assay, steric exclusion is involved, in that receptors orantibodies for the ligand and for the fluorescer are employed, wheresimultaneous binding of the receptor for the ligand and receptor for thefluorescer is inhibited. Furthermore, when the receptor for thefluorescer (antifluorescer) is bound to the fluorescer, the fluorescenceof the fluorescer is substantially diminished. Further reduction if notcomplete inhibition of fluorescence can be achieved by conjugation ofquencher to the antifluorescer. This assay is extensively described inU.S. Pat. No. 3,998,943, issued Dec. 21, 1976. The fluorescein which isemployed there may be substituted with the fluorescent compounds of thesubject invention. The assay is described in Columns 3-5 of the subjectpatent, which description is incorporated herein by reference.

Generally, the method involves combining the samples suspected ofcontaining the analyte, the conjugate of the ligand and fluorescer,anti-fluorescer, and receptor for ligand or antiligand, when ligand isthe analyte. The materials are combined in an aqueous medium at a pH inthe range of about 5 to 10, usually in the range of about 6 to 9, at atemperature in the range of about 10° to 45° C. and the fluorescencedetermined either as a rate or equilibrium mode, readings being takenwithin about 1 second to 1 hour after all materials have been combinedfor a rate mode, while for an equilibrium mode, readings may be takenfor as long as up to about 24 hours or longer.

In the next immunoassay technique, a fluorescer-quencher pair isemployed, where one of the members of the pair is conjugated to a memberof a specific binding pair, ligand and antiligand, and the otherchromophor member is bound to the same or different member of thespecific binding pair. For example, the fluorescer and the quencher maybe bound to different molecules of antiligand, so that when the twoconjugated antiligands are brought together with antigen, the fluorescerand quencher are brought within quenching distance. Alternatively, onecould bind one of the chromogens to the ligand and the other chromogento the antiligand. This assay is extensively described in U.S. Pat. No.3,996,345. The assay technique is described beginning with Col. 17 andending at Col. 23, which description is incorporated herein byreference. The ratios of chromogen to ligand and receptor is describedin Cols. 4-6, which description is incorporated herein by reference.

The assay is carried out in substantially the same manner as describedabove, except that in this assay, the fluorescer conjugates and quencherconjugates are added in conjunction with the sample and the fluorescencedetermined in comparison to an assay medium having a known amount of theanalyte.

Other techniques may also be employed with the subject compounds, suchas techniques involving heavy atom quenching as described in co-pendingapplication Ser. No. 824,576, filed Aug. 13, 1977 or other assaytechniques where a fluorescent molecule is desired which emits light ata wavelength substantially above the light emitted by fluorescentcompounds naturally present in physiological fluids or other samples tobe analyzed.

Finally, the subject conjugates may be used in conjunction with supportsas described in U.S. patent application Ser. No. 964,099, filed Nov. 24,1978. These assays are predicated upon having the fluorescer moleculeavailable in bulk solution for interaction with a signal modulator orbound to a particle, where the particle environment prevents theinteraction. Alternatively, the particle can provide an environmentwhich modulates the fluorescent signals when the fluorescer conjugate isbound to the particle.

EXPERIMENTAL

The following examples are offered by way of illustration and not by wayof limitation.

(All temperatures not otherwise indicated are centigrade. All parts andpercents not otherwise indicated are by weight, except for mixtures ofliquids which are by volume. The following abbreviations are employed.TLC--thin layer chromatography; THF--tetrahydrofuran; DCC--dicyclohexylcarbodiimide; NHS--N-hydroxy succinimide; T₃ --triiodothyronine.

EXAMPLE I Preparation of2,7-dimethyl-9-(3',4'-dicarboxy-2',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

A. 4-Methyl phthalic anhydride (20.0 g) was dissolved in 20% fumingsulphuric acid (25 ml) and powdered iodine (0.5 g) added. The mixturewas heated to 90°-100° and chlorine gas bubbled through the solutioncontinuously. After 24 hrs heating, 0.5 g more of iodine was added andthe heating continued for 24 hrs more. After 2 days of heating, a whitesolid had precipitated out. The solution was cooled and diluted in 100ml of ice-cold water and filtered to yield a white solid. The solid waswashed with cold water (20 ml) and dried in vacuo. A sample of theproduct 3,5,6-trichloro-4-methylphthalic acid and anhydride (I) washydrolyzed to yield a white powder m.p 226°-8°.

B. (I) (20 g, 0.075 moles) was placed in a 1-liter, 3-neck flaskequipped with mechanical stirrer and a water condenser and 400 ml 10% K₂CO₃ added. The slurry was refluxed in an oil bath at 120° until all thesolid had dissolved (about 1 hr). After adding 10 ml tert-butanol, 33.0g (0.2 moles) powdered KMnO₄ was added in portions to the hot stirringsolution. Care was taken to avoid accumulation of KMnO₄. An additional100 ml of 10% K₂ CO₃ was used to wash in the KMnO₄. After all KMnO₄ hadbeen added, the reaction was checked by TLC. [TLC was taken by thefollowing procedure: A sample of the reaction mixture was acidified with6M H₂ SO₄ ; the excess KMnO₄ was reacted with a saturated solution ofoxalic acid; the clear solution was extracted with ether. The ethersolution was reacted with an ether solution of diazomethane andconcentrated under N₂. TLC was taken of the methyl ester in 100%benzene; Rf=0.3. The Rf of the methyl ester of the diacid derived from Iwas 0.5.]

The reaction was stopped when all the diacid was gone by TLC. Thetert-butanol was distilled off. The stirring slurry was acidified with6M H₂ SO₄ to pH1. The excess KMnO₄ was removed by reaction with solidoxalic acid. Sulfuric acid (6M) was added to keep the mixture at pH 1during the oxalic acid addition. The solution was concentrated on aRotovap, yielding a white slurry. Hydrochloric acid (6M) was added untilthe total volume was approximately 300 ml and all the solid haddissolved. The solution was stirred for 30 min at room temperaturegiving a fine white precipitate. The slurry was extracted with ether[3×400 ml]. A white inorganic salt came out in the aqueous layer. Theether was taken off on a Rotovap. The resulting oil was azeotropicallydried using dry benzene on the Rotovap. A white powder (20 g) wasisolated. The powder was recrystallized in ethyl acetate-carbontetrachloride to give 19.5 g of 3,5,6-trichlorotrimellitic acid (II). mp238°-240°.

C. In a 250 ml R.B. flask was dissolved the trichlorotriacid (II, 10 g)in 30 ml acetic anhydride and the mixture heated at 140°-45° under N₂for 45 min. After cooling, the acetic anhydride was removed on a Rotovapunder high vacuum, while heating at 35°-40°. After complete removal ofacetic anhydride, the flask was left on high vacuum directly overnightto remove the last traces of acetic anhydride, and the product (III) wasused directly.

D. The trichloroanhydride (III, 9.5 g) was mixed with powdered4-methylresorcinol (8.5 g, dried overnight on high vacuum) in a widemouth tube and heated in a preheated oil bath at 185°-90°. AnhydrousZnCl₂ (1 g) was added to the mixture and the heating continued for 1.5hrs with occasional mixing with a spatula, a hard red mass beingobtained. The contents were cooled and the red solid scraped off of thetube.

The solid was dissolved with stirring in 300 ml of 8% aq. NaOH, cooledin an ice bath and acidified with 1:1 HCl to pH1. A yellow solidseparated out. After filtering and washing with water (150 ml), theprecipitate was dried under high vacuum overnight. The dry yellow solidweighed about 15.5 g.

The dried yellow powder was stirred with 200 ml of ethyl acetateovernight, the mixture filtered and the solids washed with 15 ml ethylacetate. The ethyl acetate filtrate was concentrated on a Rotovap at˜35°-40° to almost dryness. The residue was stirred with 200 ml benzenefor 2 hrs and filtered. The filtered solid was stirred with 200 ml ofCH₂ Cl₂ for 3-4 hrs and again filtered. The remaining solid weighed 9.0g. Its TLC in THF:CH₂ Cl₂ (1:1) indicated that it is almost pure withone major spot on TLC and a couple of minor spots moving almost with thesolvent front. The yellow solid was used without further purificationfor the next reaction.

The above yellow solid mixture (6.0 g) was dissolved in 150 ml offreshly distilled THF (distilled over CaH₂) and 3.0 g DCC added. Theclear solution was then stirred overnight. Next day, the white solidwhich had formed was filtered, the solid washed with 15 ml dry THF andthe combined filtrates concentrated to dryness on a Rotovap at ambienttemperature. To the solid was then added 200 ml n-hexane and the mixturestirred for 2 hrs to remove excess DCC. The yellow solid was filteredand washed with 50 ml n-hexane. The remaining solid is a mixture ofunreacted VI and anhydride VII as shown by TLC (solvent system THF:CH₂Cl₂ 60:40).(VI-2,7-dimethyl-9-(2',4'-dicarboxy-3',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one;VII-2,7-dimethyl-9-(3',4'-dicarboxyanhydride-2',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one).

Example II Preparation of 2,7-dimethyl-9-(3' or 4'-carboxamido-4' or3'-carboxy-2',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one aceticacid

The yellow solid obtained in Example I was dissolved in dry THF (300 ml)and combined with 8.5 g of 3-β-cholestanyl glycinate and the mixturestirred overnight at room temperature. The solvent was then removed andthe residual solid stirred with water (150 ml) for 2 hrs. The resultingmixture was acidified with dil HCl to pH1 and stirring continued for 1hr more in the cold room. The resulting yellow solid was filtered andwashed with 100 ml of ice-cold water and dried in vacuo. Its TLC(THF:CH₂ Cl₂ 1:1) indicated it to be a mixture of only two majorcompounds. The yellow solid was absorbed on silica gel (30 g) with THFand dried. The dry powder was poured over a dry column of silica gel(200 g) and eluted with THF:CH₂ Cl₂ mixture (1:4) with the elutionfollowed by TLC. The faster moving spot eluant was collected and thesolvent removed to give the cholestanyl ester derivative of the aboveindicated compound as a yellow powder (2.6 g).

Hydrolysis of the above ester was carried out by dissolving 1.5 g of theester in 10 ml of THF, adding aq. NaOH (1 g in 70 ml H₂ O) and stirringthe solution for 24 hrs at room temperature. A white solid separatedout. The alkaline solution was extracted with ether (3×50 ml) and theaq. solution acidified with conc. HCl to pH 2. A yellow solid separatedout. This solution was extracted with ether (3×100 ml), the etherealsolution washed with brine solution (2×15 ml) and upon removal of theether a yellow solid was obtained, which was dried in vacuo for 4 hrs.The residual mass was stirred with CH₂ Cl₂ (45 ml) overnight, resultingin a yellow powder precipitating. This was filtered and washed with moreCH₂ Cl₂ (20 ml) and the final yellow solid (700 mg) dried in vacuo.

EXAMPLE III Conjugation of the product of Ex. II to human IgG (a)Preparation of the activated NHS derivative of the dye

The product of Ex. II (60 mg) was dissolved in dry THF (1 ml) and NHS(30 mg) added, followed by the addition of DCC (30 mg) and stirring atroom temperature for 3 hrs. A white solid separated out which wasfiltered off. The filtrate was concentrated in vacuo and the residuestirred for 30 min with 10 ml of n-hexane to remove excess DCC. Theyellow powder was filtered off. It was used directly.

(b) Reaction of NHS ester of the dye to human IgG

Human IgG (10 mg) was dissolved in 1.2 ml of 0.05M PO₄ ³⁻ buffer at pH8.0 and cooled to 0°-5° in an ice cold bath. A solution of 1.2 mg of theabove prepared NHS ester in dry DMF (40 μl) was added during 20 min tothe rapidly stirring protein solution (The pH of the solution ismaintained at 8.0 during addition of NHS ester by adding a trace ofsolid Na₂ CO₃.) After the addition is complete, the mixture is stirredfor 1.5 hrs at room temperature and then 1 ml of 2N NH₂ OH (adjusted topH 8.1) added and the mixture stirred for 1 hr. more in the cold room.After centrifugation of the reaction mixture, the supernatant solutionwas purified through Sephadex G-25 column using 0.05M PO₄ ³⁻ buffer atpH 8.0. The faster moving conjugate was found to have λ_(max) ^(abs)521-22 nm and λ_(max) ^(emission) 537-38 nm. This particular conjugatewas found to have dye/protein ratio of 7.56 based on uv calculations.

EXAMPLE IV Conjugate of the product of Ex. II with triiodothyronine (T₃)and dextran

In general, great care was taken to avoid the exposure to light ofcompounds at all stages of the sequence.

A. T₃ (1 g) was suspended in dry methyl alcohol (15 ml) and a slowstream of hydrogen chloride gas passed through the suspension for 20min. A clear solution was obtained. The solvent was removed on a Rotovapat room temperature, and the residual white solid dried in vacuo andused directly.

B. To a mixture of the above ester (70 mg) in dry DMF (1 ml) containingtriethylamine (100 μl) was added the NHS ester of the product of Ex. II(prepared from 70 mg in accordance with the procedure described above)and the solution stirred overnight. The DMF was removed in vacuo and theresidue treated with dil.HCl resulting in a yellow solid which wasfiltered, washed with water, and dried in vacuo. Its TLC (THF:CH₂ Cl₂::40:60) indicated that it has one major spot. The material was purifiedby preparative TLC using 16 plates (20×20 cm) and the above solventsystem. The major spot was eluted with methanol and the methanol removedto yield ˜40 mg.

C. A solution of the above product (30 mg) in 1N NaOH (2 ml) was stirredat room temperature for 2 hrs. The solution was acidified with dil.HCland the yellow solid filtered and dried. The acid was further purifiedby preparative TLC (CH₂ Cl₂ :MeOH:AcOH::75:25:1) and the product elutedfrom silica gel with methanol. After removing the methanol, the residuewas dissolved in 1N NaOH, the solution filtered and acidified withdil.HCl. The resulting yellow precipitate was filtered, washed withwater and dried overnight in vacuo at 65° to give the desired acidproduct (13 mg). Its UV spectrum in 0.05M PO₄ ³⁻ buffer had λ_(max)^(absorption) 519 nm, and λ_(max) ^(emission) 533-34 nm.

D. Aminodextran was prepared from BHP activated Dextran 70 (Sigma) asdescribed in copending application Ser. No. 017,874, filed Mar. 3, 1979.BHP activated Dextran 70 (500 mg) was stirred overnight with 10 ml ofbuffer (0.15M NH₄ OH and 0.1M NaHCO₃ -Na₂ CO₃, pH 9.0) at roomtemperature. β-Mercaptoethanol (70 μl) was added and stirring continuedat room temperature for 10 hrs. After dialyzing against water at roomtemperature over the weekend, the total volume after dialysis was about14 ml (contains about 500 mg of Dextran 70).

E. A solution of 10 mg of the acid of C in dry THF (1 ml), containingDCC (9 mg) and NHS (5 mg), was stirred overnight. A white solidseparated out. After filtering, the filtrate was concentrated in vacuo.The residue was macerated with hexane (10 ml) and the yellow solid (˜20mg) separated. On TLC examination in THF:CH₂ Cl₂ (1:1), the yellow solidappeared to be mostly the NHS ester (higher Rf) with a small amount ofstarting material (lower Rf).

F. Aminodextran solution (300 μl) prepared above was diluted with 300 μlof 0.1N NaHCO₃ -Na₂ CO₃ solution (pH 9.0), and to this was added asolution of 0.5 mg of the NHS ester prepared in E in 25 μl of THF. Thesolution was stirred at room temperature for 1.5 hrs. To this solutionwas then added 0.3 ml of 3N NH₂ OH solution (pH 8.0) and the mixturestirred for 45 min at room temperature. After centrifugation for 2 min,the supernatant was purified over G-25 Sephadex column (1×30 cm) using0.1N NaHCO₃ -Na₂ CO₃ buffer (pH 9.0). The faster moving conjugate wascollected (total volume of conjugate after chromatographing is about 2ml). It had λ_(max) ^(absorption) 521-22 nm, λ_(max) ^(emission) 537-38nm. Its quantum yield was 43% as compared to the parent fluoresceinderivative of Ex. II (based on emission peak area when both are excitedat 500 nm).

EXAMPLE V Preparation of 2,7-dimethyl-9-(2',4' or 5'dicarboxyphenyl)-6-hydroxy-3H-xanthen-3-one and the 4,5-dichloroderivative

A. In a reaction flask was combined 1.35 g 4-methylresorcinol, 1 gtrimellitic anhydride and 100 mg ZnCl₂ and the mixture heated at195°-200° for 15 min. The resulting solid was macerated with water andfiltered. The precipitate was dissolved in 5% NaOH, stirred for 5 min,filtered and the filtrate acidified with dil.HCl to pH2. The resultingsolid was recrystallized from methanol to yield an orange red solid.mp>280°.

B. A portion of the above product was dissolved in DMF, and 2.2 equiv.of chlorine in glac. acetic acid added. When no further reactionappeared to be occurring, the product was isolated and purified bypreparative TLC using CHCl₃ :MeOH(80:20).

EXAMPLE VI Preparation of2,7-di(2"-carboxyethyl)-9-(2'-carboxyphenyl)-6-hydroxy-3H-xanthen-3-one

Into a reaction flask was introduced 1.1 g of4-(2'-carboxyethyl)resorcinol, 0.45 g phthalic anhydride and 250 mgZnCl₂ and the mixture heated at 160°-70° for 0.5 hr. After treating withwater, and filtering, the solid was dissolved in 5% NaOH, the solutionstirred for 15 min, the filtrate acidified with dil HCl to pH2 and theresulting yellow solid filtered and dried. The product was maceratedwith ethyl acetate and further purified by preparative TLC (CHCl₃:MeOH:CH₃ CO₂ H glac.::80:20:0.5).

EXAMPLE VII Preparation of 2,7-di(3"-carboxypropyl)-9-(2',4'-and3',4'-diicarboxy-3',5',6' and2',5',6'-trichlorophenyl)-6-hydroxy-3H-xanthen-3-one

Following the prior procedures, into a reaction flask was introduced 2.6g 4-(3'-carboxypropyl)resorcinol, 1.95 g3,5,6-trichloro-1,2,4-benzenetricarboxylic acid and 100 mg ZnCl₂ and themixture heated at 180°-85° for 40 min. The mixture was worked up aspreviously described and the product purified by preparative TLC usingCHCl₃ :MeOH:HOAc::80:20:1.

The following table reports the spectroscopic properties of thedescribed compounds.

                  TABLE III                                                       ______________________________________                                                absorption.sup.1                                                                        emission.sup.1                                              Ex.     λ.sub.max                                                                        λ.sub.max                                                                          ε × 10.sup.3.spsp.1                                                    φ.sup.2, %                           ______________________________________                                        I       516       531         70.5   76                                       II      518       532-3       72.0   70                                       V A     500       518-9       67.3   92                                       B        511-2    523-5              100                                      VI       500-1    521-2       70.0   88                                       VII     519       535         69.0   64                                       ______________________________________                                         .sup.1 In 0.05 M PO.sub.4.sup.3- buffer, pH 8.0                               .sup.2 Compared to fluorescein on the basis of emission peak measured in      0.05 M PO.sub.4.sup.3- excited at 475 nm.                                

It is evident from the previous examples that the subject compounds havemany desirable properties. The compounds absorb at wavelengths at or inexcess of 500, having large Stokes shifts, and their spectroscopicproperties are not adversely affected by being conjugated to proteins.

In order to demonstrate further the utility of the subject inventions,immunoassays were carried out, where the fluorescent compound was acompound within the scope of the subject invention. The assay wasconcerned with immunoglobulin G. The following solutions were employedin preparing reagents for carrying out the assay:

Buffer 0.01M Na₂ HPO₄, 0.15M NaCl, 2%PEG6000, 0.05% NaN₃, pH8.0.

Reagent B diluent: 0.05M trizma base, 0.02M glycine, 0.01Mbenzamine-HCl, 0.15M NaCl, 0.05% NaN₃, pH8.0.

Reagent A diluent: 0.05M trizma base, 1% cholate, 0.01M benzamidine-HCl,0.05% NaN₃, pH8.0.

Calibrator diluent: 0.01M Na₂ HPO₄, 0.15M NaCl, 0.01M benzamidine-HCl,0.01% NaN₃, pH7.2.

Reagent A is a rhodamine-anti(hIgG) conjugate diluted 1:30 with reagentA diluent and having a rhodamine/protein ratio of 11. Reagent B is a2,7-dimethyl-9-(2',5',6'-trichloro-3' or 4'-carboxamido glycine-4'- or3'-carboxy)-6-hydroxy-3H-xanthen-3-one conjugate to hIgG diluted 1:26.7with reagent B diluent and having a fluorescer/protein ratio of about 2.

The calibrators are derived from Freon-dextran sulfate treated serum,diluted with calibrator diluent, except for the 24 mg/ml calibratorwhich employes undiluted serum. The negative calibrator is assay buffer.

The assay is performed with a Varian Fluorichrom fluorometer (modified),at 25°, employing as the gain a fluorescence signal set to 1600 using astandard solution of 3.8×10⁻⁹ M of the fluorescer itself.

The assay is performed by combining 16 μl of the calibrator or sample,diluted with 400 μl of assay buffer, following by the addition of 50 μleach of reagent A and reagent B and an additional 400 μl of assaybuffer. After 5 sec, the change in fluorescence is read over a 6 secperiod, to determine the rate of change in fluorescence.

A standard curve was prepared having the following higG mg/mlconcentrations: 0, 1.6, 5.6, 11.2, 17.6 and 24.0.

A number of samples were then employed and the hIgG determined andcompared with commercially available RID or nephenlometric techniques.The following table indicates the results.

                  TABLE III                                                       ______________________________________                                                             Correlation                                              Comparative                                                                            No. of   Inter-         Coeffi-                                                                              Standard                              Method   Samples  cept     Slope cient  Error                                 ______________________________________                                        RID      50       -0.12    1.12  0.97   1.49                                  Nephelo- 25       3.87     0.72  0.91   1.76                                  metry (1)                                                                     Nephelo- 12       3.25     0.59  0.96   1.65                                  metry (2)                                                                     ______________________________________                                    

It is evident from the above results, that there is a consistentrelationship between the subject method employing the compounds of thesubject invention and other commercially available assays. There is anevident bias in the nephelometric results, possibly due to each of thetechniques employing its own method of valuation.

The subject invention provides novel compounds which have importantspectroscopic properties, providing for absorption at long wavelengths,high extinction coefficients, sharp absorption bands and fluorescentbands and substantial spacing between absorption and fluorescence bands.These properties are particularly desirable and important to thedevelopment of fluorescent techniques or the detection of a wide varietyof materials.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

What is claimed is:
 1. In a method for performing a fluorescent proteinbinding assay, which comprises combining in an assay medium a samplesuspected of containing an analyte and members of a signal producingsystem wherein one of said members comprises a conjugate of a member ofa specific binding pair either ligand or receptor and aspectroscopically active compound, and measuring the level of saidsignal in said assay medium, the improvement which comprises employingas said conjugate a compound of the formula ##STR9## wherein: α is amember of a specific binding pair either ligand or receptor covalentlybonded to one of ψ or ψ';ρ is an aliphatic hydrocarbon group of fromabout 1 to 12 carbon atoms. ψ' is hydrogen or non-oxocarbonyl; L is abond or divalent radical; ψ is a linking group bonded to α or a groupterminating in a heteroatom containing functionality when not bonded toα; and ν is a number between 1 and the molecular weight of α divided by500.
 2. In a method for performing a fluorescent protein binding assay,which comprises combining in an assay medium a sample suspected ofcontaining an analyte, and members of a signal producing system whereinone of said members comprises a conjugate of a member of a specificbinding pair either ligand or receptor and a spectroscopically activecompound, the improvement which comprises employing as said conjugate acompound of the formula ##STR10## wherein: R is alkyl or carboxyalkyl offrom 1 to 4 carbon atoms:Z is carboxy; A is a ligand or receptor; W is abond or divalent radical of from 0 to 8 carbon atoms; Y is methylene ora heteroatom containing linking functionality; m is 0 to 3; n is anumber between 1 and the molecular weight of A divided by 500; and saidportion in the brackets of said formula having from 0 to 6 chloro groupsat other than the 1,8-position of the xanthene portion of the molecule.3. In a method for performing a fluorescent protein binding assay, whichcomprises combining in an assay medium a sample suspected of containingan analyte, and members of a signal producing system wherein one of saidmembers comprises a conjugate of a member of a specific binding paireither ligand or receptor and a spectroscopically active compound, theimprovement which comprises employing as said conjugate a compound ofthe formula ##STR11## wherein: E^(b) is hydrogen or chloro;E² is chloro;Z² is carboxy; R² is alkylene of from 1 to 3 carbon atoms; D² ishydrogen or carboxy; W² is a bond, alkylene, carboxamidoalkylene, orpoly(carboxamidoalkylene), wherein alkylene is of from 1 to 2 carbonatoms; Y² is non-oxo-carbonyl, carbamoyl, thiocarbamoyl, amino,methylene, oxy or thio; A² is a ligand or receptor; m² is 2 to 3; X² is0 to 4; and n² is a number between 1 and the molecular weight of A²divided by
 500. 4. In a method for performing a fluorescent proteinbinding assay, which comprises combining in an assay medium a samplesuspected of containing an analyte, and members of a signal producingsystem wherein one of said members comprises a conjugate of a member ofa specific binding pair either ligand or receptor and aspectroscopically active compound, which conjugate is further bound to asupport, and measuring the level of said signal in said assay medium,the improvement which comprises employing as said conjugate bound to asupport a compound of the formula ##STR12## wherein: n³ is a numberbetween 1 and the molecular weight of A² divided by 500, with theproviso that when q is 0, n³ is 1;q is 0 to 1; p is at least 1 and up tothe molecular weight of Support divided by 500; Support is a molecule ofat least about 10,000 molecular weight having a plurality offunctionalities for linking; the group in the brackets is bonded by anyconvenient functionality by a bond or linking group to the Support;E^(b) is hydrogen or chloro; E² is chloro; Z² is carboxy; R² is alkyleneof from 1 to 3 carbon atoms; D² is hydrogen or carboxy; W² is a bond,alkylene, carboxyamidoalkylene or poly(carboxyamidoalkylene), whereinalkylene is of from 1 to 2 carbon atoms; Y² is non-oxo-carbonyl,carbamoyl, thiocarbamoyl, amino, methylene, oxy or thio; A² is a ligandor receptor bonded to Y² ; m² is 0 to 3; and x² is 0 to
 4. 5. In amethod for performing a fluorescent protein binding assay, whichcomprises combining in an assay medium a sample suspected of containingan analyte, and members of a signal producing system wherein one of saidmembers comprises a conjugate of a member of a specific binding paireither ligand or receptor and a spectroscopically active compound, theimprovement which comprises employing as said conjugate a compound ofthe formula ##STR13## wherein: E^(b) is hydrogen or chloro;R² isalkylene of from 1 to 2 carbon atoms; D² is hydrogen or carboxy; m² is 1to 3; x² is 1 to 3; a² is 0 to 1; A² is a ligand or a receptor, saidligand and receptor being linked by amide linkages; n² is 1 when A² ishydroxyl or an electrophilic ester activating group and is otherwise anumber between 1 and the molecular weight of A² divided by 500.